Recombinant kid pregastric esterase and methods for its production and use

ABSTRACT

The present invention provides kPGE and derivative polypeptides which are capable of being produced by genetic recombination and used to produce EMCs. This invention further provides nucleic acid sequences encoding kPGE and derivative polypeptides which can be used to create recombinant host cells that express kPGE and derivative polypeptides. A further subject of the present of invention is a fusion polypeptide called polyHis-enterokinase which increases expression of esterases and lipases when fused to the N-terminal of the esterase or lipase. This invention also provides a method for treating animals with an esterase or lipase deficiency by administering rkPGE to the animal in a therapeutically effective amount.

Throughout this specification, various references are identified by anumber in parentheses. The citation to the reference corresponding tothe identified number can be found in the section entitled ReferencesCited preceding the claims. The references in that section are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Esterases (also referred to as lipases) are enzymes that cleavetriglycerides (fats or lipids) or esters into carboxylic acids (fattyacids) and mono- and di-glycerides. For an explanation of the slightlydifferent definitions given to lipases and esterases see Siezen, R. J.and van den Berg, (37). A pregastric esterase is an esterolytic orlipolytic enzyme secreted by the oral tissues of mammals. Animalesterases in an unpurified form called rennet have been used in theproduction of dairy food products and, in particular, the production ofenzyme modified cheeses or EMCs. (8), (9), (10), (17), (18), (33), (40),and (41). In particular, cheeses like Romano and Provolone have a“peppery” or “piccante” flavor due to the fatty acid composition createdby the enzyme in the rennet paste. (26), (37).

Traditionally EMCs are prepared by esterases obtained from the gullet ofslaughtered animals from which a rennet paste or powder is obtained. Therennet is used to treat whey to impart flavor into the cheese product.Kid pregastric estersase (kPGE or kid PGE) in rennet paste iscontaminated with proteins which are found in the gullet of the kid andother substances used in the preparation of the rennet. It would beuseful to have an uncontaminated kPGE to produce EMC's. Such EMC's couldbe produced in a manner acceptable to kosher and vegetarian consumers. Arecombinant kPGE (rKPGE) could be produced in very pure form free of theother substances found in the present commercial rennet formulations.

SUMMARY OF THE INVENTION

The present invention provides kPGE and derivative polypeptides whichare capable of being produced by genetic recombination and used toproduce EMCS. This invention further provides nucleic acid sequencesencoding kPGE and derivative polypeptides which can be used to createrecombinant host cells that express kPGE and derivative polypeptides. Afurther subject of the present of invention is a fusion polypeptidecalled polyHis-enterokinase which increases expression of esterases andlipases when fused to the N-terminal of the esterase or lipase. Thisinvention also provides a method for treating animals with an esteraseor lipase deficiency by administering rkPGE to the animal in atherapeutically effective amount.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a comparison of an amino acid sequence for kid pregastricesterase to the amino acid sequences of bovine gastric esterase, humangastric lipase and rat lingual lipase.

FIGS. 2(A), 2(B), and 2(C) are a comparison of the genes encoding kidand bovine pregastric esterase.

FIG. 3 is an amino acid and codon summary for the isolated kidpregastric esterase corresponding to the N-terminal sequence.

FIG. 4 is the HPLC separation purified fragments collected as individualspecies corresponding to 210 nm absorbance peaks.

FIG. 5 is the amino acid sequence for human gastric lipase in singleletter form and homologous regions of the corresponding kPGE partialamino acid sequences are indicated.

FIG. 6 shows a method for the construction of a cDNA library used tofind the cDNA sequence for the kPGE gene.

FIG. 7 depicts an extraction device for free fatty acids.

FIG. 8 is a typical chromatogram of a free fatty acid standard.

FIG. 9 is a comparison of the Free Fatty Acid (FFA) Profile of KidPregastric Esterase (KPGE) from Lipolyzed Butter Oil comparing thecarboxylic acid mixture from lypolized butter oil using recombinant kidpregastric esterase and native kid pregastric esterase.

FIG. 10 is a schematic diagram of an expression vector (pPIC9K) with asequence encoding kPGE.

FIGS. 11(A) and 11(B) depict the procedure for expression of kPGE in thePichia expression system. FIG. 11(A) Clone the gene of interest into oneof the Pichia expression vectors. Perform a transformation of theexpression vector. Linearize the resulting construct by digestion withNot I or BgI II. Prepare sphereoplasts of his4 Pichia pastoris strainGS115 then transform the spheroplasts with the linearized construct.Recombination occurs in vivo between the 5′ and 3′ AOX1 sequences in thePichia pastoris vector and those in the genome. This results in thereplacement of the AOX1 gene with the kidPGE gene. The Pichia pastorisgenome now contains the kPGE and the HIS4 gene. Transformants are platedon histidine-deficient media. Cells in which recombinantion has occurredwill grow, others will not produce histidine and will die.

FIG. 11(B) depicts screening for a recombinant strain expressing thekPGE gene. Screen for integration at the correct loci. Select coloniesfrom the −his plaste and patch onto a −his, +glycerol and a−his,+methanol plate. Colonies which grow slowly on the −his,+met plateno longer contain the AOX1 gene and have a his+,mut− (methanolutilization deficient) phenotype. Pilot expression can now occur byselecting 10-20 his+,mut−colonies and grow for two days in mediacontaining glycerol as the carbon source. Pellet the cells and removethe media. To induce expression, resuspend pellet in media containingmethanol as the carbon source and grow the cells for 2-6 days. Analyzeprotein expression by SDS-PAGE and Western blot techniques. Based on thepilot expression results, expression can be scaled up.

FIG. 12 schematically depicts a FLAG® expression sequence for kidpregastric esterase using the polyHis-enterokinase fusion polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The term “kPGE” refers to kid pregastric esterase. The term “rkPGE”refers to recombinant kid pregastric esterase. Kid pregastric esteraseincludes alleles of naturally occurring kid pregastric esterase. KPGE isan enzyme that is capable of producing a carboylic acid mixture from inabout the same mixture as a commercial kid rennet preparation. Apolypeptide derivative of kPGE is capable of the same function as kPGEbut differs in the amino acid sequence of kPGE in at least one of theways described below.

Derivatives of kPGE can differ from naturally occurring kPGE in aminoacid sequence or in ways that do not involve sequence, or both.Derivatives in amino acid sequence are produced when one or more aminoacids in naturally occurring kPGE is substituted with a differentnatural amino acid, an amino acid derivative or non-native amino acid.Particularly preferred embodiments include naturally occurring kPGE, orbiologically active fragments of naturally occurring kPGE, whosesequences differ from the wild type sequence by one or more conservativeamino acid substitutions, which typically have minimal influence on thesecondary structure and hydrophobic nature of the protein or peptide.Derivatives may also have sequences which differ by one or morenon-conservative amino acid substitutions, deletions or insertions whichdo not abolish the kPGE biological activity. Conservative substitutions(substituents) typically include the substitution of one amino acid foranother with similar characteristics such as substitutions within thefollowing groups: valine, glycine; glycine, alanine; valine, isoleucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. The non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

Other conservative substitutions can be taken from Table 1, and yetothers are described by Dayhoff in the Atlas of Protein Sequence andStructure (1988).

TABLE 1 Conservative Amino Acid Replacements For Amino Acid Code Replacewith any of Alanine A D-Ala, Gly,beta-ALa, L-Cys,D-Cys Arginine R D-Arg,Lys,homo-Arg, D-homo-Arg, Met,D-Met, Ile, D-Ile, Orn, D-Orn Asparagine ND-Asn,Asp,D-Asp,Glu,D-Glu, Gln,D-Gln Aspartic Acid D D-Asp,D-Asn,Asn,Glu,D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys,Met,D-Met,Thr, D-ThrGlutamine Q D-Gln,Asn, D-Asn,Glu,D-Glu,Asp, D-Asp Glutamic Acid ED-Glu,D-Asp,Asp, Asn, D-Asn, Gln,D-Gln Glycine G Ala, D-Ala,Pro, D-Pro,Beta-Ala,Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met,D-MetLeucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys,Arg, D-Arg,homo-Arg, D-homo- Arg, Met, D-Met, Ile, D-Ile, Orn,D-Orn Methionine MD-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val, NorleuPhenylalanine F D-Phe,Tyr, D-Thr,L-Dopa,His,D-His, Trp, D-Trp, Trans 3,4or 5- phenylproline, cis 3,4 or 5 phenylproline Proline P D-Pro,L-I-thioazolidine-4- carboxylic acid, D- or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met,D-Met, Met(O), D-Met(O), Val, D-Val Threonine T D-Thr, Ser, D-Ser,allo-Thr, Met, D-Met, Met(O) D-Met(O), Val, D-Val Tyrosine Y D-Tyr,Phe,D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu,D-Leu,Ile,D-Ile, Met, D-Met

Other derivatives within the invention are those with modificationswhich increase peptide stability. Such derivatives may contain, forexample, one or more non-peptide bonds (which replace the peptide bonds)in the peptide sequence. Also included are: derivatives that includeresidues other than naturally occurring L-amino acids, such as D-aminoacids or non-naturally occurring or synthetic amino acids such as betaor gamma amino acids and cyclic derivatives. Incorporation of D-insteadof L-amino acids into the polypeptide may increase its resistance toproteases. See, e.g., U.S. Pat. No. 5,219,990, incorporated by referenceherein.

The polypeptides of this invention may also be modified by variouschanges such as insertions, deletions and substitutions, eitherconservative or nonconservative where such changes might provide forcertain advantages in their use.

In other embodiments, derivatives with amino acid substitutions whichare less conservative may also result in desired derivatives, e.g., bycausing changes in charge, conformation and other biological properties.Such substitutions would include for example, substitution ofhydrophilic residue for a hydrophobic residue, substitution of acysteine or proline for another residue, substitution of a residuehaving a small side chain for a residue having a bulky side chain orsubstitution of a residue having a net positive charge for a residuehaving a net negative charge. When the result of a given substitutioncannot be predicted with certainty, the derivatives may be readilyassayed according to the methods disclosed herein to determine thepresence or absence of the desired characteristics.

Derivatives within the scope of the invention include proteins andpeptides with amino acid sequences having at least eighty percenthomology with kPGE. More preferably the sequence homology is at leastninety percent, or at least ninety-five percent.

Just as it is possible to replace substituents of the scaffold, it isalso possible to substitute functional groups which decorate thescaffold with groups characterized by similar features. Thesesubstitutions will initially be conservative, i.e., the replacementgroup will have approximately the same size, shape, hydrophobicity andcharge as the original group. Non-sequence modifications may include,for example, in vivo or in vitro chemical derivatization of portions ofnaturally occurring kPGE, as well as changes in acetylation,methylation, phosphorylation, carboxylation or glycosylation.

In a further embodiment the protein is modified by chemicalmodifications in which activity is preserved. For example, the proteinsmay be amidated, sulfated, singly or multiply halogenated, alkylated,carboxylated, or phosphorylated. The protein may also be singly ormultiply acylated, such as with an acetyl group, with a farnesyl moiety,or with a fatty acid, which may be saturated, monounsaturated orpolyunsaturated. The fatty acid may also be singly or multiplyfluorinated. The invention also includes methionine analogs of theprotein, for example the methionine sulfone and methionine sulfoxideanalogs. The invention also includes salts of the proteins, such asammonium salts, including alkyl or aryl ammonium salts, sulfate,hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate,thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate,mesylate, ethyl sulfonate and benzensulfonate salts.

Derivatives of kPGE may also include peptidomimetics of kPGE. Suchcompounds are well known to those of skill in the art and are producedthrough the substitution of certain R groups or amino acids in theprotein with non-physiological, non-natural replacements. Suchsubstitutions may increase the stability of such compound beyond that ofthe naturally occurring compound.

A yeast strain comprising a recombinant DNA molecule which expresses kidpregastric esterase was deposited with the Northern Regional ResearchCenter and received deposit no. NRRL Y-30030.

It will be appreciated from the present disclosure that the kidpregastric esterase and derivatives and fatty acid mixtures according tothe present invention can be used to alter, vary, fortify modify,enhance or otherwise improve the taste of a wide variety of materialswhich are ingested, consumed or otherwise organoleptically sensed.

The terms “alter” and “modify” in their various forms will be understoodherein to mean the supplying or imparting of a flavor character or noteto an otherwise bland, relatively tasteless substance, or augmenting anexisting flavor characteristic where the natural flavor is deficient insome regard or supplementing the existing flavor impression to modifyits organoleptic character.

The term “enhance” is intended herein to mean the intensification (bythe use of the kid pregastric esterase and derivatives of the presentinvention) of a flavor or aroma note or nuance in a foodstuff or dairyproduct or cheese without changing the quality of said note or nuance.

The term “flavoring composition” is taken to mean one which contributesa part of the overall flavor impression by supplementing or fortifying anatural or artificial flavor in a material or one which suppliessubstantially all the flavor and/or aroma character to a consumablearticle.

The term “foodstuff” as used herein includes both solid and liquidingestible materials for man or animals which materials usually do, butneed not, have nutritional value. Thus, foodstuffs include meats,gravies, soups, convenience foods, malt, alcoholic, milk and dairyproducts, seafoods, candies, vegetables, animal foods, veterinaryproducts and the like. The kid pregastric esterase and derivatives ofthe present invention are useful in the creation of flavor in cheeses orcheesefoods or any other food containing triglycerides.

The carboxylic acid mixture produced by the kid pregastric esterase andderivatives of the present invention can be combined with conventionalflavoring agents or adjuvants. Such co-ingredients or flavor adjuvantsare well known in the art for such and have been extensively describedin the literature. Requirements of such adjuvants are: (1) that they benon-reactive with the carboxylic acid mixture of the present invention;(2) that they be organoleptically compatible with the mixture of thepresent invention such that the flavor of the mixture is not adverselyaffected by the use of the adjuvant; and (3) that they be ingestiblyacceptable and thus non-toxic or otherwise non-deleterious. Appart fromthese requirements, conventional materials can be used and broadlyinclude other flavor materials, vehicles, stabilizers, thickeners,surface active agents, conditioners, and flavor intensifiers.

The following terms are used in accordance with their meanings in theart. DNA is deoxyribonucleic acid whether single- or double-stranded.Complementary DNA (cDNA) is DNA which has a nucleic acid sequenceobtained from reverse transcription of messenger ribonucleic acid(mRNA). Recombinant genetic expression refers to the methods by which aDNA molecule encoding a polypeptide of interest is used to transform ahost cell so that the host cell will express the polypeptide ofinterest. A plasmid or vector can be used to introduce a DNA moleculeinto a host cell. A plasmid or vector can comprise, but need not, inaddition to the gene or nucleic acid sequence of interest, a gene thatexpresses a selectable marker or phenotype and a gene that can control(induce or inhibit) the expression of the gene of interest under certainconditions.

This invention comprises a kid pregastric esterase which is free ofother kid proteins. The kPGE can be produced by purifying the kidpregastric esterase from kid gullet or by recombinant genetic expressionin a non-kid cell. The non-kid cell can be a bacterial, a fungal, ayeast or an animal cell. In a preferred embodiment, the yeast isSaccharomyces cerevisiae. The bacterial cell E. Coli can be used as canthe Chinese Hamster Ovary cell. In the invention, the kid pregastricesterase have glycosylation which is different than that of kidpregastric esterase produced in a kid cell.

The present invention further provides a polypeptide comprising an aminoacid sequence addition, substitution, or deletion derivative of kidpregastric esterase wherein the polypeptide is capable of convertingfats to fatty acids in about the same ratio as kid pregastric esteraseis capable of converting. The ratio of fatty acids the polypeptidederivative is capable of converting has about the same flavor as would aratio of fatty acids converted by kid pregastric esterase and the fatscapable of being converted are from a dairy product. In one embodiment,a polyHis-enterokinase is added to the N-terminal of the amino acidsequence of kid pregastric esterase. The polyHis-enterokinase can havethe amino acid sequence in SEQ. ID. NO. 6.

The invention further provides a polyHis-enterokinase polypeptide. Thispolypeptide is capable of increasing lipase polypeptide expression whenexpressed at the N-terminal of the lipase polypeptide. In a furtherembodiment, the polyHis-enterokinase polypeptide comprises at least 5His amino acids and can comprise the amino acid sequence in SEQ. ID. NO.6.

The present invention provides isolated polynucleotides capable ofexpressing the polypeptides of the present invention. In one embodiment,the polynucleotide encodes an amino acid sequence of kid pregastricesterase or a derivative polypeptide of kPGE or a polypeptide which iscomplementary to the nucleic acid sequence of SEQ. ID. NO. 1. Thepolynucleotide can be DNA or RNA. The polynucleotide can comprise anucleotide sequence encoding a polyHis-enterokinase polypeptide. In afurther embodiment, the polynucleotide comprises the nucleic acidsequence of SEQ. ID. NO. 7.

The present invention provides a transforming nucleic acid moleculecomprising a plasmid or vector comprising a nucleic acid sequenceencoding the amino acid sequence of kid pregastric esterase or aderivative polypeptide. The transforming nucleic acid can comprise thenucleic acid sequence of SEQ. ID. NO. 5.

The present invention further provides cell capable of recombinantlyexpressing kid pregastric esterase or a polypeptide derivative of kPGE,wherein the cell has been tranformed with the nucleic acid encoding theexpressable polypeptide. The cell can be a bacterial, a fungal, a yeastor an animal cell. In a preferred embodiment, the cell is the yeast cellSaccharomyces cerevisiae.

The present invention also provides a monoclonal antibody to thepolypeptides of the subject invention.

The present invention discloses a process for recombinantly producingkid pregastric esterase by isolating a polynucleotide encoding an aminoacid sequence for kid pregastric esterase; inserting the isolatedpolynucleotide into a vector or plasmid suitable to transform a hostcell; transforming a host cell with the vector or plasmid comprising theisolated polynucleotide; and growing the transformed cells to expresskid pregastric esterase.

The present invention teaches a method of treating an esterase deficientanimal, wherein the animal is treated by administering a therapeuticallyeffective amount of the kid pregastric esterase or derivative. In anembodiment, a pharmaceutical composition comprising a pharmaceuticalcarrier and a therapeutically effective amount of the kid pregastricesterase or a derivative.

The present invention discloses a mixture of fatty acids produced byreacting kid pregastric esterase with a dairy product. The dairy productcomprises lipolyzed butter oil, milk, cheese or whey. The presentinvention further discloses a process for producing a mixture of fattyacids comprising reacting a dairy product with a kid pregastricesterase. Thus, the kid pregastric esterase of the present invention iscapable of being used in the production of EMC's as a substitute for acommercial rennet preparation and may be used in addition to such apreparation as well.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details Ultra Purification of kPGE Protein

A 250 ml 2.5×50 cm Bio-Rad Econo-column was packed with approximately220 ml Bio-Rad Exchange Q chromotrography matrix as specified bysupplier. The column was washed (200 mls) and equilibrated in 50 mMTris-Cl, pH 8.0 (=buffer).

Five grams of Aurotech Kid Pregastric Esterase (390 Ramsey Units) werebrought up in 50 mM Tris-Cl, pH 8.0, mixed by stirring for approximately20 min and centrifuged at 6500 rpm in a GAS rotor (7000 g) for 10 min.The supernatent was decanted and recentrifuged as before. Eighty mls wasrecovered and loaded into the column running at 2 mls/min.

The column was washed with buffer for 100 min. Initially the flow ratewas 2 mls/min but this changed to approximately 1.5 mls/min by the endof this period. At 100 min, a 100 min linear gradient from 100% A=bufferto 100% B=1 M NaCl, 50 mM Tris-Cl, pH 8.0, was begun. Twenty fiveminutes into gradient, collection of fractions (2 min=3 mls) were begunand continued for 124 min. At 200 min, the gradient was held at 100% Bfor 50 min, they switched to 100% A and held 200 min to re-equilibratethe column.

Activity of the kPGE was assayed at 405 nm using p-nitrophenol buteratesubstrate. Twenty μl samples or dilutions are placed in microtiter dishwells and diluted with 180 μl of substrate solution prepared as follows:thirty mg of p-nitrophenol buterate is dissolved in 10 mls isopropanoland 1 ml added to 9 mls of 4.4% Triton X-100, 0.11% Gum Arabic, 50 mMTris-Cl, pH 8 solution.

Fractions containing kPGE activity were pooled, diafiltered with 20 mMBIS-TRIS (bis[2-Hydrosyethyl]imino-tris[hydrosymethyl] methane) buffer,pH 7.1, and loaded onto a column containing 200 ml of PBE 94chromatography gel (Pharmacia Biotech, Inc., Piscataway, N.J.) forchromatofocusing in the pH range of 9-4. The column was developed with a1 to 10 dilution of Polbuffer 74 (Pharmacia Biotech, Inc., Piscataway,N.J.), pH 4.0. Fractions were collected and assayed at 405 nm usingp-nitrophenol buterate substrate as described above. Fractionscontaining kPGE activity were pooled, concentrated and diafilteredagainst distilled water using a stirred cell device (Amicon, Inc.,Beverly, Mass.), fitted with a high-flow, inert non-ionic membraneretaining 90% of molecules with molecular masses greater than 30,000Daltons, i.e. PM30 (Amicon, Inc., Beverly, Mass.

Partially-purified and concentrated kPGE from the chromatofocusingcolumn was subjected to electrophoresis in precast 12% polyacrylamidegels containing 375 mM Tris-Cl buffer, pH 8.8 (Bio-Rad Laboratories,Hercules, Calif.) to separate protein species from one another.Following separation, the kPGE protein species was localized to specificregion of the gel by making horizontal cuts (˜1 mm segments) along thelength of the gel. This resulted in a continuous series of ˜1 mmsegments that contained protein species that had migrated at similarrates to end up in the same relative position in the gel. A small pieceof each individual segment was macerated in Tris-Cl buffer, pH 8, andassayed for activity using p-nitrophenol buterate substrate as describedabove. Those acrylamide segments showing PGE activity were thenmacerated in buffer and subjected to electrophoresis in anelectroelution devise (Isco, Inc. Lincoln Nebr.). In this manner, PGEactivity was electroeluted and concentrated in buffer. PGE activity wasreconfirmed using the p-nitrophenol buterate assay and electrophoresedin sodium dodecyl sulfate (SDS) to demonstrate recovery of an ˜50,000Dalton protein species. In addition, traditional Ramsey unit assays wereconducted to verify that classical pregastric esterase (i.e. lipase)activity was recovered. The assay procedure follows the rate of changein pH that results from lipase acting on tributerin to release butyricacid. Combined lots of this ultra-purified kPGE were assayed forfunctionality.

Functionality Verification

Purified kPGE was assayed for a determination of lipolytic activity onmilk butter fat and functionality evaluation in flavor modification.

Determination of Partial Amino Acid Sequences of kPGE and Demonstrationof Homology of These Sequences to Other Preduodenal Lipase Enzymes

Following native gel electrophoresis, proteins in the polyacrylamide gelwere electrophoretically transferred to a polyvinylidenedifloride (PVDF)membrane support using the Western blot procedure. This procedureinvolves layering the gel between filter paper and immersing the entiregel in a tank filled with a buffer solution containing 25 mM Tris, 192mM Glycine and 20% methanol. Two large electrodes on either side of thefilter paper-gel-PVDF membrane sandwich allow horizontal electrophoretictransfer of the proteins in the gel to the PVDF membrane. Followingtransfer, brief staining of the PVDF membrane with Commassie BrilliantBlue R-250 (0.025% Commassie R-250 in 40% methanol; destained with 50%methanol) allowed recognition of the ultra-purified kPGE protein as aunique band. This unique protein band of ultra-purified kPGE wasprecisely trimmed from the PVDF membrane and subjected to N-terminalamino acid sequencing procedures to yield a partial N-terminal sequence.Multiple recoveries of similarly purified kPGE bands on PVDF supportswere also subjected to protease digestion to release specific kPGEpeptide fragments. The resulting fragment mixture was then subjected toHPLC separation, see FIG. 4, and the separated, purified fragmentscollected as individual species corresponding to 210 nm absorbancepeaks. Individual fragment species were then subjected to N-terminalamino acid sequencing to obtain sequence data for three additionalfragments internal to the kPGE protein. These sequences are presented inFIG. 3 along with corresponding potential DNA codons that can prescribethe amino acids in these peptide sequences.

A search of the NBRF protein database using these partial amino acidsequences led to the identification of high homology with regions of thehuman gastric lipase and rat lingual lipase. The amino acid sequence forhuman gastric lipase is shown in single letter form in FIG. 5 wherehomologous regions of the corresponding kPGE partial amino acidsequences are indicated.

Isolation of mRNA and Construction of cDNA Library of Cloned Sequencesfrom Kid Lingual Tissue

Frozen kid pregastric tissues, i.e. oral tissues known to producelipolytic or esterolytic enzymatic activity, were homogenized in a lysisbuffer (Tris-Cl, pH 8.0, LiCl, EDTA, Li dodecyl sulfate anddithiothreitol) polyadenylated messenger RNA (polyA-mRNA) isolated usinga commercial product, Dynabeads Oligo (dT)25 (Dynal, Inc., Lake Success,N.Y.). In particular, polyA-mRNA was isolated from the parotid salivaryglands and sublingual tissues of kid tongue, but other pregastrictissues for the lipolytic or esterolytic activity of interest as well.Purified polyA-mRNA was primed with an oligonucleotide consistingprimarily of poly-deoxythymidine DNA and reverse transcribed into DNAusing reverse transcriptase using procedures analogous to those outlinedin FIG. 6. The resulting double-stranded DNA molecules were then cutwith Eco R1 restriction enzyme and ligated into Eco R1-cut Lambda ZAP IIvector DNA (Stratagene Cloning Systems, La Jolla, Calif.) to produce alibrary of Lambda ZAP II DNAs, each of which presumably contained onecDNA derived from one mRNA that was present in kid lingual tissue mRNApopulation. The library of cDNA-containing Lambda ZAP II DNAs was thenpackaged to form virulent bacteriophage using a commercially preparedpackaging extract (Gigapack II gold packaging extract, StratageneCloning Systems, La Jolla, Calif.) and used to infect an appropriatestrain of bacteria (XL1-Blue, Stratagene Cloning Systems, La Jolla,Calif.). Each infected cell contains only one type of Lambda ZAP IIphage which replicates within the cell, directs the production ofpackaging components and becomes packaged to release many virulent phage(all identical in DNA structure) upon lysis of the cell. A primary phagelibrary thus results that contains millions of virulent phage thatcomprise a population of phage, may of which contain cDNAs.

Development of Oligonucleotide Probes for Recognition and Recovery ofthe kPGE Gene from the cDNA Library

From the kPGE amino acid sequences determined above, syntheticoligonucleotides were designed to be used in generating fragments of DNAthat represent parts of the kPGE gene. Certain regions of the partialamino acid sequences were reverse translated into corresponding DNAsequences that would act as primers for DNA synthesis in the polymerasechain reaction (PCR). PCR techniques allow synthesis and amplificationof regions of DNA that lie between two oligonucelotides (primers), oneof which hybridizes to the plus strand and the other that hybridizes tothe minus strand. Several rounds of synthesis lead to the generation ofmany copies of the fragment of DNA that lies between the two primers.The sequence of nucleotides found in the primers provides thespecificity of the region of DNA that will be amplified. Since specificamino acids can be specified by more than one codon, mixtures ofsynthetic oligonucleotides were prepared that contained representativesof all possible sequences for the region of interest. Theseoligonucleotides were used in conjunction with similar oligonucleotidesdeveloped from conserved and homologous regions of similar enzymes, i.e.human gastric lipase, to synthesize segments of the kPGE gene using thePCR reaction. Since enzymes of this type, i.e. preduodenal lipases, areremarkably similar in size, ˜50,000 Daltons, and the relative regions ofhomology of the partial kPGE amino acid sequences were known, therelative size of the expected DNA fragment from PCR synthesis with anytwo appropriate primers could be predicted. Thus, specific kPGE-basedprimers, conserved lipase-based primers or combinations thereof wereused to carryout PCR using the library of cDNA-containing bacteriophageto generate specific DNA fragments. Several of these combinationsyielded DNA fragment sizes expected to result from authentic kPGE genesequences, but did not yield correctly sized DNA fragments if only thevector, i.e. Lambda ZAP II (not containing cDNA), DNA was used. In thisway, the library was shown to contain DNA sequences of kPGE-like genes.DNA fragments generated from these PCR amplifications were cloned into aplasmid vector, pT7Blue T-vector (Novagen, Inc., Madison, Wis.) andtransformed into bacterial cells (Novablue competent E. Coli fromNovagen, Inc., Madison, Wis.) using well-known bacterial transformationprocedures. Each transformed cell, i.e. clone, contained many copies ofone type of plasmid which contained a DNA fragment corresponding to asegment of a kPGE-like gene. Plasmid DNA preparations were made fromseveral different transformed clones to recover larger quantities ofpurified DNAs containing different kPGE-like gene fragments. Several ofthese were DNA sequenced using common techniques and one (from clone GS1972) was selected as clearly containing DNA sequence that whentranslated would produce a protein with very high amino acid sequencehomology to comparable regions of other preduodenal and lingual lipases.

Identification and Recovery of the Cloned kPGE Gene From the cDNALibrary

Plasmid DNA from a bacterial clone, i.e. GS 1972, was shown to consistof plasmid vector, pT7Blue T-vector (Novagen, Inc., Madison, Wis.), withan integrated PCR-generated DNA fragment (441 basepairs) correspondingto the translated region of amino acid residue ˜18 to ˜164 of otherknown, mature preduodenal and lingual lipases. This purified DNA wasradioactively labeled with S35 by common procedures and used to identifyphage carrying cDNAs with homologous regions by common screeningprocedures. Since the primary phage library contains millions of phagein a highly concentrated form, several rounds of phage purification mustbe conducted to separate the phage of interest, i.e. those containingkid-PGE-like cDNAs, from all others. Thus, semi-purified phagepreparations were first identified by diluting the phage and planting onagar such that single phage plaques, i.e. a population of phage derivedfrom only one phage, were clearly identified. Replicas of the phageplaque patterns that occurred on agar plates were then transferred tonitrocellulose membranes and probed with the radioactively labelledprobe by common procedures to identify phage plaques of interest. Phagewere then taken from the positive plaques on the agar plates and used toidentify those that yielded an ˜440 basepair fragment when amplifiedusing the original primers in PCR experiments. Following initialidentification of 10 positive semi-purified phage preparations,secondary and tertiary screens were performed as above to result in theidentification of 5 highly-purified phage preparations that yielded ahybridizing signal when labeled with radioactive plasmid DNA from cloneGS 1972 and an ˜440 basepair fragment when amplified using the originalprimers in PCR experiments.

These 5 highly-purified phage preparations were then used to infect XL1Blue cells (Stratagene Cloning Systems, La Jolla, Calif.) along with M13helper phage to convert the cloned fragments from a phage form into aplasmid form, i.e. a phagemid. Proteins produced by the M13 helper phagecut the phage DNA on one side of the cloned insert DNA and replicate theDNA through to the other side. This smaller newly synthesizedsingle-stranded DNA is then circularized, packaged and secreted from thecell. The secreted phagemid is then used to transform SOLR bacterialcells (Stratagene Cloning Systems, La Jolla, Calif.) along with anotherhelper phage, VCSM13, to convert the phagemid into a replicating, stableplasmid. The SOLR cells are designed to prevent the replication of bothM13 and Lambda phage such that only cells containing replicatingplasmids are recovered when plated on an ampicillin-containing agarplate. In this way, 4 E. Coli strains were obtained that containedpBluescript SK-doublestranded phagemids with cloned cDNA inserts ofinterest. DNA sequencing of the cDNA inserts of these phagemids, yieldeda nucleotide sequence, a portion of which translated into a PGE-likeenzyme. The translated sequence is comprised of 378 amino acids thatform a protein with a calculated molecular mass of 42,687 Daltons. Bycomparison, human gastric lipase is comprised of 378 amino acids and hasa calculated molecular mass of 43,208 Daltons; bovine pregastricesterase is comprised of 378 amino acids and has a calculated molecularmass of 42,987 Dalatons; while rat lingual lipase is comprised of 376amino acids and has a calculated molecular mass of 42,700 Daltons. Acomparison of the amino acid sequence alignments indicates thesimilarity among these enzyme, FIG. 1. At the DNA level, strong homologyis still quite apparent, FIGS. 2(A-C). Inspection of the translatedsequence of the PGE-like gene confirmed the presence of amino acidsequences that were determined above from the purified kPGE enzyme, thusconfirming recovery of the kPGE gene. Expression of the kPGE inmicroorganisms and transgenic animals is possible with the nucleic acidsequence which can be used in recombinant genetic expression. Arecombinant kPGE can fixed and delivered into food systems by spraydrying or encapsulation. This kPGE, as the result of controlledsynthesis and recovery of a highly purified form with naturallipase/esterase activity, is likely to be used to create new dairyflavors. Microbial production will allow the development of new Kosherand vegetarian food products.

Esterase Functionality Assay

All esterase samples were received frozen and stored in ˜18° C. freezer.Before being used, they were thawed and stored in 5° C. refrigerator.Original kid lipase, lot #81882, 390 U/gram samples: PGE SAMPLE 1; 0.6ml at 5 U/ml in 20 mM phosphate buffer at pH 7.0; PGE SAMPLE 2: 0.6 mlat 5 U/ml in 20 mM phosphate buffer at pH 7.0; and PGE CONTROL: 0.6 mlin 20 mM phosphate buffer at pH 7.0. The substrate was 40% fat creamobtained from Golden Guemsey Dairy. Cream is free of added mono anddiglyeride. All chemical reagents were obtained from Aldrich ChemicalCompany, Inc. and were the best grade available.

Usage level of all lipase samples are 0.78 U/gram of cream, which iscomparable to the usage level in production. Samples are received in acapped 10 ml plastic tube. According to the usage level, 3.8 gram ofcream is added into each tube. For the control, the lipase powder isdissolved in 20 mM phosphate buffer at pH 7.0 to the activity level of 5Ramsey units per milliliter. Then, 0.6 ml of the solution is mixed with3.8 grams of cream substrate in a capped plastic tube. They areincubated at 37° C. for 72 hours. At the end of incubation, no heat isapplied because of small sample size. All samples are stored in arefrigerator until analysis.

Analysis

Titration

Each sample was titrated at the end of incubation. Because of thesmaller sample size, titration could not be used to follow the fattyacid development. Take 0.1 gram sample from the flask and dissolve thesample with 50 ml isopropanol. Add 2-3 drops of phenolphthaleinindicator and titrate it with 0.05 N NaOH till end point. Record the mlof 0.05 N NaOH used and convert to per gram basis.

Free Fatty Acid Profile

Free fatty acid profile of each sample was analyzed using the procedurebelow. This method quantify the following fatty acids: butyric,hexanoic, octanoic, decanoic, lauric, myristic, palmitic, palmitoleic,stearic, oleic, linoleic and linolenic acids. Results are expressed asmole percentage of the total free fatty acid. Because we did not haveduplication due to limitation of enzyme samples, each sample wasanalyzed twice and results were averaged.

Organoleptic Analysis

At the end of incubation, all enzyme samples have a different titration.A cheese sauce consisting of margarine, modified starch, and Velveetacheese and water was used as the base for evaluation because it has beenroutinely used for evaluation of such samples. Since the samples had adifferent titration, the usage level of each on is varied to compensatefor the titration variations.

RESULTS

Total free fatty acids released by each enzyme preparation were quitedifferent, even though the same amount of activity units were used inthe incubation. The control lipase has the highest activity.

SAMPLE TITRATION (0.05 N NaOH/g) Lipase control 5.4 PGE Sample 1 3.3 PGESample 2 2.4 PGE Control 1.7

Unfortunately we did not have a plain cream control. It is possible thatmajority of the PGE control titration is from the milk lipase existingin the cream.

Free fatty acid profiles: The PGE Control shows a typical profile ofmilk lipase. The PGE sample 1 and the Lipase control have almostidentical profiles. PGE sample 2 shows a different profile with muchlower percentage in short chain fatty acids and higher percentage inlong chain fatty acids. The overall activity of this sample is also muchlower. The change of profile could be due to the impact of milk lipasein the system. When the lipase activity is low, impact of milk lipasecould play a much bigger role. This might explain why the PGE sample 2shows a different profile.

Organoleptic results: Because each sample has different titration,percentage of sample used in the cheese sauce for organolepticevaluation varied depending on the sample strength:

SAMPLE PERCENTAGE USED Lipase Control 2.0 PGE Sample 1 3.27 PGE Sample 24.50 PGE Control 6.35

Overall, all these samples showed very similar organoleptic properties.They are not identified as typical fatty acid and had a culture milktype of flavor.

TABLE 3 Free fatty acid profile of lipase fractions FFA CONTROL PGE-S1PGE-S2 PGE-CONTROL C4:0 46.70 44.97 35.30 19.56 C6:0 15.50 15.19 13.018.75 C8:0 4.45 3.96 3.82 2.66 C10:0 8.52 7.59 7.41 5.62 C12:0 5.60 5.165.00 4.71 C14:0 5.34 5.93 7.09 10.74 C16:0 5.55 8.42 13.63 23.88 C16:11.11 1.00 1.69 2.93 C18:0 2.58 2.48 3.83 6.06 C18:1 3.26 4.57 8.18 13.50C18:2 0.90 0.58 0.87 1.44 C18:3 0.41 0.15 0.18 0.15

Assay Procedure for Lipase Activity

The substrate used is: 475 mL deionized water, 45 mL Tributyrin, 3 gSodium Caseinate and 2.5 g Lecithin blended in a Waring blender. The pHwas adjusted to 5.5 with 88% Lactic Acid and temperature 42° C.

An enzyme standard was created: For Concentrate:  1 gram Standard in 99mL of 3% NaCl; and For Dilute: 10 gram Standard in 90 mL of 3% NaCl

stirred in a tempered water bath at 42C for 15 min. The kid reference(400 R.U.'s) standard is 1:100 while the calf reference (68 R.U.'s)standard is 1:10.

Fill a 50 mL buret on a ring stand with 0.05N NaOH. Place 100 mL ofsubstrate into a 250 Ml beaker and immerse a standardized pH electrodeinto the beaker. Place the beaker and electrode on a magnetic stir plateset at 3.8 to heat the substrate to 42° C. while stirring constantly andadjust the pH to 5.5. Dispense 10 mL of Enzyme Standard Solution intothe 250 mL beaker. Set the timer for 6 minutes and adjust the flow ofNaOH to retain the pH at 5.5. Keep track of the amount of NaOH used thelast 5 min. After 6 minutes has elapsed, close the buret and record theamount consumed.

Calculation

Calculate activity according to the following formulas:

Concentrate 1/100 Sample 1/100 (Kid) Sample 1/100 of Conc. Kid 1/10 ofCut Kid

(R.U.'s of control/Titer of control)×Titer of sample=Sample R.U.

Concentrate 1/100 Sample 1/10 (CALF)

(R.U.'s of control/Titer of control)×Titer of sample×1/10=Sample R.U.

Procedure for Extraction and Analysis of Free Fatty Acids From Lipolizedand EMC Products

This procedure extracts free fatty acids from lipolized butter andenzyme modified cheeses. The extract is then analyzed by gaschromatography method. This procedure is adapted from Deeth, H. C. etal. “A gas chromatography method for the quantitative determination offree fatty acid in milk and milk products” New Zealand Journal of DairyScience and Technology, 18:13-20, which is hereby incorporated byreference. This procedure has been extensively tested for extractionefficiency. The adjustment for this procedure is the sample size whichdepends on the amount of free fatty acids in the sample. In Deeth etal., a packed GC column is used without further esterification. Here abonded phase capillary tube is used to give a superior chromotogramcompared to Deeth et al., especially for long chain fatty acids.Heptanoic acid is used as an internal standard for fatty acids withchain length of up to 10 carbons, while pentadecanoic acid is used forfatty acids with chain length of more than 12 carbons. In theory, onecould use only one standard, for example heptanoic acid to do thecalibration. The two internal standards used were chosen because verylittle of them exists in dairy products. The following reagents areused: necessary free fatty acids, isopropyl ether 99%, diethyl ether99.9% (spectrophotometric grade), hexane (spectrophotometric grade),formic acid 96% (ACS reagent), activated aluminum oxide (acidic,Brockman I), 4N sulfuric acid, and glass wool treated with phosphoricacid.

This procedure uses a three level calibration for each fatty acid peak.However, it cannot quantify acetic acid in the product because formicacid used in the procedure contains small amounts of acetic acid whichinterferes with quantifying the acetic acid extracted from the sample.

Weigh the following amount of fatty acids into a 100 ml volumetric flaskdirectly:

TABLE 4 Fatty Acid Levels LEVEL 1 in LEVEL 2 in LEVEL 3 in FATTY ACIDSmg mg mg propionic acid 50 50 50 butyric acid 50 50 50 caproic acid 5050 50 heptanoic acid 60 40 20 (ISTD) caprylic acid 50 50 50 capric acid50 50 50 lauric acid 50 50 50 myristic acid 50 50 50 pentadecanoic(ISTD) 60 40 20 palrritic acid 50 50 50 palmitoleic acid 50 50 50stearic acid 50 50 50 oleic acid 50 50 50 linoleic acid 50 50 50linolenic acid 50 50 50

Use an analytical scale to weigh each compound and record to 0.1 mg. Itis recommended that one starts weighing standards from the longer chainlength fatty acids because they are less volatile. This will minimizeloss due to evaporation. FIG. 8 shows a typical chromatogram of thestandard. Note that retention time will vary depending on total lengthand column and condition of the column. Add 4 gram of formic acid intoeach flask. Fill each flask to mark with isopropyl either and mix wellbut exercise care to prevent leaking during mixing. Standard can bestored in small glass bottles (˜10 ml) with Teflon® or other chemicalresistant cap in freezer for future uses.

Preparation of Internal Standard (ISTD) Solution

Weigh about 0.3 gram of heptanoic acid and pentadecanoic acid into a 100ml volumetric flask. Use and analytical scale for the weighing andrecord weight to 0.1 mg. Fill the flask with hexane till mark. Be surethe hexane is at room temperature. Mix well but exercise care to preventleaking during mixing. Store internal standard solution in small glassbottles (˜10 ml) with Teflon® or other chemical resistant cap inrefrigerator for future uses. When it is to be used, be sure to let thebottle warm up to room temperature. Leaving the bottle at roomtemperature overnight is recommended.

Preparation of Column Packing

Deactivated alumina is used for adsorption of free fatty acids from theextract. Activated aluminum oxide, acidic, Brockmann I is used. It isdeactivated according to the following steps. Preheat a drying oven to225° C. Place about 20 grams alumina in a 100 ml beaker. Cover beakerwith aluminum foil and make some holes on the foil with sharp needle.Place it into the heated oven. Each extraction uses about 1 gram ofalumina. Dry it at this temperature for 2 hours. Stop heating of oven.Remove beaker and cool it in desiccator. Transfer to small wide-mouthbottle with screw cap. Close cap tightly. Record the powder dry weight.After dry powder is cool, add 4% water to deactivate the dry powder onthe powder dry weight. Add water in four equal portions. Mix it verywell with stainless steel spatula each time water is added until mixtureis homogeneous. During this process, heat is released and the bottleshould be warm. Cap it tightly and shake the bottle for 5 minutes. Storeit in desiccator and equilibrate it at least overnight before using.

Extraction of Free Fatty Acids from Samples

This procedure is suitable for extracting free fatty acid from dairyproducts. The only difference for different samples will be the samplesize. For samples with low degree of lipolysis, e.g., sample with about4 ml 0.05 N NaOH free fatty acid titration per gram, use about 0.3 gramsample. For sample with strong lipolysis, such sample with free fattyacid titration of 16 ml 0.05 N NaOH per gram, use about 0.1 gram samplesize. Caution: all extraction work has to be done in hood with goodventilation.

Make a solvent mix for extraction. It contains hexane/diethyl either atration of 1:1 (vol:vol). Make enough to finish all extraction (about35-40 ml for extraction). Weigh sample into a 50 ml centrifuge tube withscrew cap. Caution: selection of centrifuge tube is very importantbecause we are centrifuging ether at very high speed. Selecting thewrong type of centrifuge tube might create danger. Nelgene FEP oak ridgecentrifuge tube with ETFE sealing cap assembly is selected because ofits superior chemical compatibility. Add 0.1 ml of 4 N sulfuric acidinto each tube. Add 0.100 ml of ISTD solution into each tube. Use a 0.2ml glass pipet in 0.01 ml graduation to deliver the ISTD solution. Donot piper the solution with mouth. Calculate mg of internal standardcompound added per gram of sample. This value is used for calculationduring analysis according to the following formula:$\text{mg ISTD/g sample} = \frac{0.100*\text{ISTD conc. in mg/ml}}{\text{sample size in grams.}}$

Add 1 gram of anhydrous sodium sulfate into each tube. Add 10 ml of theextraction solvent form step 1 into each tube. Cap the tube tightly andmix it well with Vortex mixer at highest setting. Often the sample willdissolve in the extraction mixture. Extract it for 30 minutes to 1 hour.For lipolized butter samples which dissolve in extraction mixtureeasily, a 30 minute extraction is enough. For enzyme modified cheeses,extract it for about an hour. Make sure there is no lumping, use a smallspatula to break any lumps.

Centrifuge the tubes at 7000 rpm at 0° C. using Sorval SA-600 rotor for5 minutes to obtain a clear supernatent which contains free fatty acidsand fat. Be sure all the tubes are balanced before startingcentrifugation. Pack a glass column with deactivated alumina. To dothat, insert a small plug of acid treated glass wool into bottom of a 30cm×11 mm chromatography column and than pack it with 1 gram of thedeactivated alumina. Set up the column on a filter flask as shown inFIG. 7.

Carefully pour the supernatent from the centrifugation into a smallglass beaker. Slowly introduce it into the column. Adjust the flow rateso that it drips slowly (about 0.5-1 drop/second). After all the extractpasses through the column, wash the small beaker with liquid collectedin the filter flask and pass it through the column again. Wash thecolumn with 5 ml hexane/diethyl either mixture twice at the same flowrate as at (about 0.5-1 drop/second). When introducing the solventmixture into column, pour it slowly along the side of column so thesolvent washes the column.

Start the vacuum slowly so when a finger is placed at the end of thevacuum hose, you can barely feel the suction. Connect the hose to filterflask to dry the alumina. Dry it till no lumping occurs when you tiltthe column and the packing inside appears to be free flowing powder.Transfer the alumina into a small vial and cap tightly. Remove the glasswool from column. Wash the whole set up with extraction mixture so itwill be ready for next sample. Column and flask can be blow-dried withair.

Prepare at 6.0% formic acid solution with isopropyl either. This is usedto release fatty acid in the alumina packing. Weight 0.5 gram aluminafrom step 12 into a disposable microcentrifuge tube (1.5 ml capacity).Add 0.5 ml formic acid solution from step 14. Cap it and mix thorough.Let stand for abut 30 minutes with occasional mixing. Centrifuge it withMICROSPIN 12 centrifuge for 2 minutes to obtain clear supernatent.Transfer the supernatent into a 1 ml vial and cap it. This supernatentis ready for GC analysis.

Analysis of the Extract

The free fatty acid extract was analyzed by gas chromatograph (GC)method, using the following conditions:

GC: HP5890 II Column: HP-FFAP,25 M × 32 mm with 0.52 um film thicknessGuard Column: Restek capillary guard column, 5 M × 0.32 mm InjectorTemperature: 28 C. Detector Temperature 300 C. Oven Temperature: 100C.-240 C. at 8 C./min. Initial Isothermal Time: 0 Minutes FinalIsothermal Time: 12.5 minutes Total Analysis Time: 30 minutes InitialInlet Pressure: 20.0 psi Constant Flow: On Flow Rate: 3.7 ml/min SplitFlow: 20.0 ml/min. Detection: FID Injection: 0.2 ul

Stabilwax-DA 30 M×0.32 mm with 0.25 um film thickness from Restek can bealso used with slightly less satisfaction for fatty acids with 18 carbonchain length.

Calculation of the Fatty Acids (mg) Per Gram of Product

The three levels of standard are analyzed with the same GC program. Acalibration table is built containing three level linear calibration foreach peak. After sample is analyzed, a report of mg/ml sample will beprinted.

Calculating the Mol % of Fatty Acids Per Gram of Product

To calculate the Mol % (Mol % is more useful for recognizing a fattyacid profile): calculate the mmole of each fatty acid per gram sample.To do that, divide the result of each fatty acid (unit: mg/g sample) byits molecular weight. Sum all the calculated mmole of each fatty acidper gram sample to obtain the total mmole of free fatty acid per gram ofsample. Divide the mmole of each fatty acid per gram sample by the totalmmole of free fatty acid per gram of sample. Multiple the calculatedvalue by 100. This gives you the Mol %. To verify the calcualtion, thesum of the Mol % results of all fatty acids should total 100.

Expression Systems

General Characteristics of Pichia pastoris

The yeast Pichia pastoris, a microbial eukaryote, has been developedinto a premier expression system. As a yeast, Pichia pastoris is as easyto use as E. coli, while having the advantages of eukaryotic expression(e.g. protein processing, folding, and posttranslational modifications).While possessing these advantages, it is faster, easier, and cheaper touse than other eukaryotic expression systems, such as baculovirus ormammalian tissue culture, and generally gives higher expression levels.P. pastoris is similar to the baker's yeast, Saccharomyces cerevisiae,including having the advantages of molecular and genetic manipulations,but with the added advantages of 10- to 100-fold higher heterologousprotein expression levels and the protein processing characteristics ofhigher eukaryotes.

Pichia pastoris is completely amenable to the genetic, biochemical, andmolecular biological techniques that have been developed over the pastseveral decades for S. cerevisiae with little or no modification. Inparticular, transformation by complementation, gene disruption and genereplacement techniquest developed for S. cerevisiae work equally wellfor Pichia pastoris.

The genetic nomenclature adopted for Pichia pastoris mirrors that usedfor S. cerevisiae (unlike that of Sc. pombe). For example, the gene fromS. cerevisiae that encodes the enzyme histidinol dehydrogenase is calledthe HIS4 gene and likewise the homologous gene from Pichia pastoris thatencodes the same enzyme is called the Pichia pastoris HIS4 gene, and soon. There is a very high degree of cross-functionality between Pichiapastoris and S. cerevisiae. For instance, many S. cerevisiae genes havebeen shown to genetically complement the comparable mutants in Pichiapastoris, and vice versa (e.g. the Pichia pastoris HIS4 genefunctionally complements S. cerevisiae his4 mutants and the S.cerevisiae HIS4 gene functionally complements Pichia pastoris his4mutants; other cross-complementing genes that have been identifiedinclude LEU2, ARG4, TRP1, and URA3).

Pichia pastoris as a Methylotropic Yeast Pichia pastoris, representingone of four different genera of methylotropic yeasts, which also includeCandida, Hansenula, and Torulopsis, is capable of metabolizing methanolas a sole carbon source. The first step in the metabolism of methanol isthe oxidation of methanol to formaldehyde by the enzyme alcoholoxidase.Expression of this enzyme, coded for by the AOX1 gene, is tightlyregulated and induced by methanol to very high levels, typically ≧30% ofthe total soluble protein in cells grown with methanol as the carbonsource. The AOX1 gene has been isolated and a plasmid-borne version ofthe AOX1 promoter is used to drive expression of the gene of interestfor heterologous protein expression.

Expression of the AOX1 gene is controlled at the level of transcription.IN methanol grown cells approximately 5% of the polyA+ RNA is from theAOX1 gene. The regulation of the AOX1 gene is similar to the regulationof the GAL1 gene (and others) of S. cerevisiae in that control involvesboth a repression/derepression mechanism. However, unlike the situationin S. cerevisiae, derepression alone of the AOX1 gene (i.e. absence of arepressing carbon source such as glucose) is not sufficient to generateeven minute levels of expression from the AOX1 gene. The inducer,methanol, is necessary for expression.

Use for Heterologous Protein Expression

Pichia pastoris has been used successfully to express a wide range ofheterologous proteins. Heterologous expression in Pichia pastoris can beeither intracellular or secreted. Secretion requires the presence of asignal sequence on the expressed protein to target it to the secretorypathway. While several different secretion signal sequences have beenused successfully, including the native secretion signal present on someheterologous proteins, success has been variable. To improve the chancesfor success, two different vectors with different secretion signals areincluded in this kit: The vector, pHIL-S1, carries a native Pichiapastoris signal from the acid phosphatase gene, PHO1. The vector, pPIC9,carries the secretion signal from the S. cerevisiae mating factorpre-pro peptide.

Another advantage of expressing secreted proteins is that Pichiapastoris secretes very low levels of native proteins that, combined withthe very low amount of protein in the Pichia growth media, means thatthe secreted heterologous protein comprises the vast majority of thetotal protein in the media and serves as the first step in purificationof the protein. Like S. cerevisiae, linear DNA can generate stabletransformants of Pichia pastoris via homologous recombination betweenthe transforming DNA and regions of homology within the genome. Suchintegrants show extreme stability in the absence of selective pressureeven when present as multiple copies.

The expression vectors included in this kit carry the HIS4 gene forselection and are designed to be linearized with a restriction enzymesuch that HIS⁺ recombinants can be generated by integration at the his4locus (a non-deletion, very low spontaneous reversion mutation) or atthe AOX1 locus. Integration events at the AOX1 locus can result in thecomplete removal of the AOX1 coding region (i.e. gene replacement) thatin turn results in a recombinant phenotype of His⁺ Mut⁻ (Mut⁻ refers tothe methanol utilization minus phenotype caused by the loss of alcoholoxidase activity encoded by the AOX1 gene that results in a no growth orslow growth phenotype on methanol media). His⁺ transformants can bereadily and easily screened for the Mut⁻ phenotype, indicatingintegration at the AOX1 locus. The His⁺ Mut⁻ clones can be furtherscreened for expression of the heterologous protein of interest.

A number of independently isolated His⁺ Mut⁻ recombinants are routinelyscreened for expression of the heterologous protein of interest becauseof the observation of clonal variation (or difference in levels ofexpressing heterologous protein seen among different transformants withthe same phenotype (His⁺ Mut⁻)). In some cases this clonal variation canbe explained by a difference in the number of copies of the integratedplasmid (i.e. more copies=more expressed protein), but it is not simplycopy number that determines protein expression level. There are severalexamples where one or more copies of the integrants express at the samelevel (and that level is high), as well as examples where an increase inthe integrant copy number causes a decrease in the protein expressionlevel, the best method at this time is to identify a successfullyexpressing clone among several (10-20) His⁺ Mut⁻ transformantsempirically.

Some examples of heterologous protein expression include:

Expression Where Protein (g/L) Expressed Reference Human serum albumin4.0 S Barr, et al (HSA) (1992) β-galactosidase 20,000 (U/mg I Tschopp,et al total protein) (1987a) Hepatitis B surface 0.4 I Cregg, et alantigen (HBSAg) (1987) Tumor Necrosis 10.0 I Sreekrishna, et Factor(TNF) al (1988) Invertase 2.3 S Tschopp, et al (1987b) Bovine Iysozymec2 0.55 S Digan, et al (1989) Tetanus toxin 12.0 I Clare, et al fragmentC (1991a) Pertusis antigen 3.0 I Romanus, et al P69 (1991) Streptokinase0.08 I Hagenson, et al (active) (1989) Human EGF 0.5 S Cregg, et al(1993) Mouse EGF 0.45 S Claire, et al (1991b) Aprotinin 0.8 S Vedvick,et al (1991) Kunitz protease 1.0 S Wagner, et al inhibitor (1992) S =secreted; I = intracellular

REFERENCES CITED

1. Anderson, R. A. and Sando, G. N. “Cloning and Expression of cDNAEncoding Human Lysosomal Acid Lipase/Cholesteryl Ester Hydrolase.Similarities to Gastric and Lingual Lipases.” J. Biol. Chem.266:22740-84 (1991);

2. Bernback, Stefan et al. “Purification and Molecular Characterizationof Bovine Pregastric Lipase” Eur. J. Biochem. 148:233-238 (1985);

3. Benicourt, Clause et al. “Acides Nucleiques Codant Pour la LipaseGastrique de Lapin et Derives Polypeptidiques, Leur Utilisation Pour LaProduction de Ces Polypeptides, et Compositions Pharmaceutiques a Basede Ces Derniers” EP 542,629 dated May 19, 1993;

4. Birschbach, Peter “Pregastric Lipases” Bulletin of the IDF 269:36-39;

5. Blanchard, Claire et al. “Recombinant Canine Gastric Lipase andPharmaceutical Compositions” WO 94/13816 dated Jun. 24, 1994;

6. Brockerhoff, H. “Determination of the Positional Distribution ofFatty Acids in Glycerolipids” General Analytical Methods 315-325;

7. Carriere, F. et al. “Purification and Biochemical Characterization ofDog Gastric Lipase” Eur. J. Biochem. 202:75-83 (1991);

8. Chapter 12 “Hard Italian Cheeses” Cheese and Fermented Milk Foods213-227;

9. Chapter 2.12, “Flavor Production with Enzymes,” IndustrialEnzymology, 2d Ed., Godfrey and West Eds. (Stockton Press, 1996);

10. Chaudhari, R. V. and Richardson, G. E. “Lamb Gastric Lipase andProteases in Cheese Manufacture” Journal of Dairy Science 54:467-71;

11. Crabbe, Thomas et al. “The Secretion of Active Recombinant HumanGastric Lipase by Saccharymoses cerevisiae” Protein Expression andPurification, 7:229-236 (1996);

12. “Current Protocols in Molecular Biology”, John Wiley & Sons, Inc,1998 (ISBN 0-471-50338-X).

13. De Laborde de Monpesat, Thierry et al. “A Fluorimetric Method forMeasuring Lipase Activity Based on Umbelliferyl Ester” ChemicalAbstracts 114:278;

14. Doeherty, A. J. P. et al. “Molecular Cloning and Nucleotide Sequenceof Rat Lingual Lipase cDNA” Nucleic Acids Res. 13:1891-1903 (1985);

15. D'Souza, Trevor M. and Oriel, Patrick “Purification andCharacterization of Lamb Pregastric Lipase” Applied Biochemistry andBiotechnology 36:183-198 (1992);

16. Eastman Kodak Company “Yeast N-Terminal FLAG® Expression System”FLAG Biosystem 1994;

17. Food Chemicals Codex, (National Academy Press, Washington, D.C.,1981) pp. 480, 493;

18. Fox, P. F. and Law, J. “Enzymology of Cheese Ripening” FoodBiotechnology 5:239-262 (1991);

19. Ha, J. Kim and Lindsay, R. C. “Influence of a_(w) on Volatile FreeFatty Acids during Storage of Cheese Bases Lipolyzed by Kid GoatPregastric Lipase” Int. Dairy Journal 2:179-193 (1992);

20. Ha, J. Kim and Lindsay, R. C. “Release of Volatile Branched-Chainand Other Fatty Acids From Ruminant Milk Fats by Various Lipases”Chemical Abstracts 118:865-66 (1993);

21. Hamosh, Margit “Lingual and Gastric Lipases” Nutrition 6:421-428(1990);

22. Komaromy, M. C. and Schotz, M. C. “Cloning of Rat Hepatic LipasecDNA: Evidence For A Lipase Gene Family” PNAS USA 84:1626-630 (1987);

23. Kurihara, Yoshie et al. “Curculin B and DNA encoding Same, andProcess for Production Thereof” AU-B-11415/92 dated Sep. 9, 1992;

24. Lowe, P. A. “New Gastric Lipase Protein, esp. of Human Origin forTreating Lipase Deficiency, and DNA Sequences Coding for It” WO/86/01532dated Mar. 13, 1986;

25. Moreau, H. et al. “Purification, Characterization and KineticProperties of the Rabbit Gastric Lipase” Biochemical et Biophysica Acta960:286-293 (1988);

26. Nelson, J. H. et al. “Pregastric Esterase and Other Oral Lipases-AReview” Journal of Dairy Science 60:327-362 (1976);

27. Parry, R. M., Jr. et al. “Rapid and Sensitive Assay for Milk Lipase”Journal of Dairy Science 49:356-360;

28. Invitrogen Corp. “Pichia Expression Kit: Protein Expression” Version3.0, Catalog No. K1710-01;

29. Invitrogen Corp. “pPIC9K A Pichia Vector for Multicopy Integrationand Secreted Expression” Version A, Catalog No. V175-20;

30. Ramsey, Harold A. “Electrophoretic Separation of Esterases Presentin Various Tissues of the Calf” Journal of Dairy Science 1185-86;

31. Ramsey, Harold A. “Photometric Procedure for Determining EsteraseActivity” Clinical Chemistry 3:185-194;

32. Ramsey, H. A. and Young, J. W. “Substrate Specificity of PregastricEsterase from the Calf” Journal of Dairy Science 2304-2306;

33. Richardson, G. H. et al. “Gastric Lipase Characterization andUtilization in Cheese Manufacture” Journal of Dairy Science 54:643-647;

34. Richardson, G. H. and Nelson, J. H. “Assay and Characterization ofPregastric Esterase” Journal of Dairy Science 50:1061-1065;

35. Sambrook, J. et al. “Molecular Cloning: A Laboratory Manual,” (ColdSpring Harbor, 1989);

36. Scorer, Carol A. et al. “Rapid Selection Using G418 of High CopyNumber Transformants of Pichia pastoris for High-level Foreign GeneExpression,” BioTechnology 12:181 (Feb. 12, 1994);

37. Siezen, R. J. and van den Berg, G. “Lipases and Their Action onMilkfat” Bulletin of the IDF 294:4-6;

38. Sweet, B. J. et al. “Purification and Charaterization of PregastricEsterase from Calf” Archives of Biochemistry and Biophysics 234:144-150(1984);

39. Talhoun, M. K. and Abdel-Ghaffar, M. “A Modified Colormetric Methodfor Assay of Lipase Activity” Chemical Abstracts 106:272;

40. Timmermans, M. Y. J. et al. “The cDNA Sequence Encoding BovinePregastric Esterase,” Gene 147: 259-262 (1994);

41. U.S. Pat. No. 2,531,329 for “Cheese Modifying Enzyme Product”(issued Nov. 21, 1950);

42. U.S. Pat. No. 2,794,743 for “Enzyme-containing Powder andEnzyme-Modified Product Thereof”;

43. U.S. Pat. No. 3,081,225 for “Enzyme Treatment for scours inanimals”;

44. U.S. Pat. No. 3,256,150 for “Method for Treating MalabsorptionSyndrome”;

45. U.S. Pat. No. 5,320,959 for “Liquid Lipase From Animal Origin andMethod of Preparation” (issued Jun. 14, 1994);

46. U.S. Pat. No. 5,521,088 for “Alcohol Acetyltransferase Genes and UseThereof” (issued May 28, 1996);

47. U.S. Pat. No. 5,529,917 for “Compositions and Methods For MakingLipolytic Enzymes” (issued Jun. 25, 1996);

48. U.S. Pat. No. 5,372,941 for “Liquid Lipase From Animal Origin”(issued Dec. 13, 1994);

49. U.S. Pat. No. 5,691,181 for “DNA Encoding Lipase From Human GastricMucosal Tissue” (issued Nov. 25, 1997);

50. U.S. Pat. No. 5,728,412 for “Alcohol Acetyltransferase Genes and UseThereof” (issued Mar. 17, 1998); and

51. Vorderwulbecke et al. “Comparison of Lipases by Different Assays”Enzyme Microb. Technol. 14:631-39 (1992).

7 1 1134 DNA Kid (Goat) CDS (1)..(1134) 1 ttc ctt gga aaa att gct aagaac cct gaa gcc agt atg aat gtg agt 48 Phe Leu Gly Lys Ile Ala Lys AsnPro Glu Ala Ser Met Asn Val Ser 1 5 10 15 cag atg att tcc ttc tgg ggctac cca agt gag atg cat aaa gtt ata 96 Gln Met Ile Ser Phe Trp Gly TyrPro Ser Glu Met His Lys Val Ile 20 25 30 act gca gat ggc tat atc ctt caggtc tat cgg att cct cat gga aag 144 Thr Ala Asp Gly Tyr Ile Leu Gln ValTyr Arg Ile Pro His Gly Lys 35 40 45 aat gat gct aat cat tta ggt cag agacct gtt gtg ttt ctg cag cat 192 Asn Asp Ala Asn His Leu Gly Gln Arg ProVal Val Phe Leu Gln His 50 55 60 ggt ctt ctt gcc tca gct aca aac tgg atttcc aac ctt ccc aac aac 240 Gly Leu Leu Ala Ser Ala Thr Asn Trp Ile SerAsn Leu Pro Asn Asn 65 70 75 80 agc ctg ggc ttc ctc ctg gca gat gct ggttat gac gtg tgg ctg ggg 288 Ser Leu Gly Phe Leu Leu Ala Asp Ala Gly TyrAsp Val Trp Leu Gly 85 90 95 aac agc aga gga aac act tgg gcc cag gaa cattta tac tat tca cca 336 Asn Ser Arg Gly Asn Thr Trp Ala Gln Glu His LeuTyr Tyr Ser Pro 100 105 110 gac tcc cct gaa ttc tgg gct ttc agc ttt gatgaa atg gct gaa tat 384 Asp Ser Pro Glu Phe Trp Ala Phe Ser Phe Asp GluMet Ala Glu Tyr 115 120 125 gac ctt cca tct aca att gat ttc atc tta aagaga aca gga cag aag 432 Asp Leu Pro Ser Thr Ile Asp Phe Ile Leu Lys ArgThr Gly Gln Lys 130 135 140 aag cta cac tat gtt ggc cat tcc caa ggc accacc att ggt ttt gtc 480 Lys Leu His Tyr Val Gly His Ser Gln Gly Thr ThrIle Gly Phe Val 145 150 155 160 gcc ttt tct acc aat ccc aca ctg gct gaaaaa atc gaa gtc ttc cat 528 Ala Phe Ser Thr Asn Pro Thr Leu Ala Glu LysIle Glu Val Phe His 165 170 175 gca tta gcc cca gtc gcc aca gtg aag cacacc cag agc ctg ttt aac 576 Ala Leu Ala Pro Val Ala Thr Val Lys His ThrGln Ser Leu Phe Asn 180 185 190 aaa ctt gca ctt att cct cac ttc ctc ttcaag att ata ttt ggt aac 624 Lys Leu Ala Leu Ile Pro His Phe Leu Phe LysIle Ile Phe Gly Asn 195 200 205 aaa atg ttc tac cca cac aat ttt ttt gaacaa ttt ctt ggt gtt gaa 672 Lys Met Phe Tyr Pro His Asn Phe Phe Glu GlnPhe Leu Gly Val Glu 210 215 220 gtg tgc tct cgt gag aca ctg gat gtc ctttgt aag aat gcc ttg ttt 720 Val Cys Ser Arg Glu Thr Leu Asp Val Leu CysLys Asn Ala Leu Phe 225 230 235 240 gcc att act gga gct gac aat aaa aacttc aac atg agt cgc tta gat 768 Ala Ile Thr Gly Ala Asp Asn Lys Asn PheAsn Met Ser Arg Leu Asp 245 250 255 gtg tat gta gca cat aat cca gca ggagct tct gtt caa aac atc ctc 816 Val Tyr Val Ala His Asn Pro Ala Gly AlaSer Val Gln Asn Ile Leu 260 265 270 cac tgg aga cag gct att aag tct gggaaa ttc caa gct ttt gac tgg 864 His Trp Arg Gln Ala Ile Lys Ser Gly LysPhe Gln Ala Phe Asp Trp 275 280 285 gga gcc tca gtt gag aac cta atg cattat aat cag ccc aca cct ccc 912 Gly Ala Ser Val Glu Asn Leu Met His TyrAsn Gln Pro Thr Pro Pro 290 295 300 atc tac aat tta aca gcc atg aat gtccca att gca gta tgg agt gct 960 Ile Tyr Asn Leu Thr Ala Met Asn Val ProIle Ala Val Trp Ser Ala 305 310 315 320 ggc caa gac ctg ttg gct gac cctcag gat gtt gac ctt ttg ctt tca 1008 Gly Gln Asp Leu Leu Ala Asp Pro GlnAsp Val Asp Leu Leu Leu Ser 325 330 335 aaa ctc tct aat ctc att cac cacaag gaa att cca aat tac aat cat 1056 Lys Leu Ser Asn Leu Ile His His LysGlu Ile Pro Asn Tyr Asn His 340 345 350 ctg gac ttt atc tgg gca atg gatgca cct caa gaa gtt tac aat gaa 1104 Leu Asp Phe Ile Trp Ala Met Asp AlaPro Gln Glu Val Tyr Asn Glu 355 360 365 att att tct ttg atg gca aaa gacaaa aag 1134 Ile Ile Ser Leu Met Ala Lys Asp Lys Lys 370 375 2 378 PRTKid (Goat) 2 Phe Leu Gly Lys Ile Ala Lys Asn Pro Glu Ala Ser Met Asn ValSer 1 5 10 15 Gln Met Ile Ser Phe Trp Gly Tyr Pro Ser Glu Met His LysVal Ile 20 25 30 Thr Ala Asp Gly Tyr Ile Leu Gln Val Tyr Arg Ile Pro HisGly Lys 35 40 45 Asn Asp Ala Asn His Leu Gly Gln Arg Pro Val Val Phe LeuGln His 50 55 60 Gly Leu Leu Ala Ser Ala Thr Asn Trp Ile Ser Asn Leu ProAsn Asn 65 70 75 80 Ser Leu Gly Phe Leu Leu Ala Asp Ala Gly Tyr Asp ValTrp Leu Gly 85 90 95 Asn Ser Arg Gly Asn Thr Trp Ala Gln Glu His Leu TyrTyr Ser Pro 100 105 110 Asp Ser Pro Glu Phe Trp Ala Phe Ser Phe Asp GluMet Ala Glu Tyr 115 120 125 Asp Leu Pro Ser Thr Ile Asp Phe Ile Leu LysArg Thr Gly Gln Lys 130 135 140 Lys Leu His Tyr Val Gly His Ser Gln GlyThr Thr Ile Gly Phe Val 145 150 155 160 Ala Phe Ser Thr Asn Pro Thr LeuAla Glu Lys Ile Glu Val Phe His 165 170 175 Ala Leu Ala Pro Val Ala ThrVal Lys His Thr Gln Ser Leu Phe Asn 180 185 190 Lys Leu Ala Leu Ile ProHis Phe Leu Phe Lys Ile Ile Phe Gly Asn 195 200 205 Lys Met Phe Tyr ProHis Asn Phe Phe Glu Gln Phe Leu Gly Val Glu 210 215 220 Val Cys Ser ArgGlu Thr Leu Asp Val Leu Cys Lys Asn Ala Leu Phe 225 230 235 240 Ala IleThr Gly Ala Asp Asn Lys Asn Phe Asn Met Ser Arg Leu Asp 245 250 255 ValTyr Val Ala His Asn Pro Ala Gly Ala Ser Val Gln Asn Ile Leu 260 265 270His Trp Arg Gln Ala Ile Lys Ser Gly Lys Phe Gln Ala Phe Asp Trp 275 280285 Gly Ala Ser Val Glu Asn Leu Met His Tyr Asn Gln Pro Thr Pro Pro 290295 300 Ile Tyr Asn Leu Thr Ala Met Asn Val Pro Ile Ala Val Trp Ser Ala305 310 315 320 Gly Gln Asp Leu Leu Ala Asp Pro Gln Asp Val Asp Leu LeuLeu Ser 325 330 335 Lys Leu Ser Asn Leu Ile His His Lys Glu Ile Pro AsnTyr Asn His 340 345 350 Leu Asp Phe Ile Trp Ala Met Asp Ala Pro Gln GluVal Tyr Asn Glu 355 360 365 Ile Ile Ser Leu Met Ala Lys Asp Lys Lys 370375 3 1411 DNA Kid (Goat) 3 gaattcggca cgagttttca tttaccttcg agaaactagaaggcattcac tttggtgaca 60 attgaaaatg tggtggctac ttgtaacggt gtgtttcatccacatgtctg gaaatgcatt 120 ttgtttcctt ggaaaaattg ctaagaaccc tgaagccagtatgaatgtga gtcagatgat 180 ttccttctgg ggctacccaa gtgagatgca taaagttataactgcagatg gctatatcct 240 tcaggtctat cggattcctc atggaaagaa tgatgctaatcatttaggtc agagacctgt 300 tgtgtttctg cagcatggtc ttcttgcctc agctacaaactggatttcca accttcccaa 360 caacagcctg ggcttcctcc tggcagatgc tggttatgacgtgtggctgg ggaacagcag 420 aggaaacact tgggcccagg aacatttata ctattcaccagactcccctg aattctgggc 480 tttcagcttt gatgaaatgg ctgaatatga ccttccatctacaattgatt tcatcttaaa 540 gagaacagga cagaagaagc tacactatgt tggccattcccaaggcacca ccattggttt 600 tgtcgccttt tctaccaatc ccacactggc tgaaaaaatcgaagtcttcc atgcattagc 660 cccagtcgcc acagtgaagc acacccagag cctgtttaacaaacttgcac ttattcctca 720 cttcctcttc aagattatat ttggtaacaa aatgttctacccacacaatt tttttgaaca 780 atttcttggt gttgaagtgt gctctcgtga gacactggatgtcctttgta agaatgcctt 840 gtttgccatt actggagctg acaataaaaa cttcaacatgagtcgcttag atgtgtatgt 900 agcacataat ccagcaggag cttctgttca aaacatcctccactggagac aggctattaa 960 gtctgggaaa ttccaagctt ttgactgggg agcctcagttgagaacctaa tgcattataa 1020 tcagcccaca cctcccatct acaatttaac agccatgaatgtcccaattg cagtatggag 1080 tgctggccaa gacctgttgg ctgaccctca ggatgttgaccttttgcttt caaaactctc 1140 taatctcatt caccacaagg aaattccaaa ttacaatcatctggacttta tctgggcaat 1200 ggatgcacct caagaagttt acaatgaaat tatttctttgatggcaaaag acaaaaagta 1260 gttctggatt tagagaatta ttcatttact ttttccaaaatagtttcttc tcacctacat 1320 gatttctgta ctgttataaa cgcaatgctt ctttctgtaatgttgacttt caaaatatat 1380 tagcatcaac aaaaaaactc gtgccgaatt c 1411 41134 DNA Bovine 4 ttccttggaa aaattgctaa gaaccctgaa gccagtatga atgttagtcagatgatttcc 60 tactggggct acccaagtga gatgcataaa gttataactg cggatggttatatccttcag 120 gtctatcgga ttcctcatgg aaagaataat gctaatcatt taggtcagagacctgttgtg 180 tttctgcagc atggtcttct tggatcagcc acaaactgga tttccaacctgcccaagaac 240 agcctgggct tcctcctggc agatgctggt tatgacgtgt ggctggggaacagcagagga 300 aacacctggg cccaggaaca tttatactat tcaccagact ccccggaattctgggctttc 360 agctttgatg aaatggcgga atatgacctt ccatctacaa ttgatttcatcttaaggaga 420 acaggacaga agaagctaca ctatgttggc cattcccaag gcaccaccattggttttatc 480 gccttttcta ccagtcccac attggctgaa aaaatcaaag tcttctatgcattagcccca 540 gttgccacag tgaagtacac caagagcctg tttaacaaac ttgcacttattcctcacttc 600 ctcttcaaga ttatatttgg tgacaaaatg ttctacccac acacttttttggaacaattt 660 cttggtgttg aaatgtgctc ccgtgagaca ctggatgtcc tttgtaagaatgccttgttt 720 gccattactg gagttgacaa taaaaacttc aacatgagtc gcttagatgtgtatatagca 780 cataatccag caggaacttc tgttcaaaac accctccact ggagacaggctgttaagtct 840 gggaaattcc aagcttttga ctggggagcc ccatatcaga acctaatgcattatcatcag 900 cccacacctc ccatctacaa tttaacagcc atgaatgtcc caattgcagtatggagtgct 960 gacaatgacc tgttggctga ccctcaggat gttgactttc tgctttcaaaactctctaat 1020 ctcatttacc acaaggaaat tccaaattac aatcacttgg actttatctgggcaatggat 1080 gcacctcaag aagtttacaa tgaaattgtt tctttgatgg ccgaagacaaaaag 1134 5 8324 DNA Yeast YE-1 expression vector 5 gatccttcaatatgcgcaca tacgctgtta tgttcaaggt cccttcgttt aagaacgaaa 60 gcggtcttccttttgaggga tgtttcaagt tgttcaaatc tatcaaattt gcaaatcccc 120 agtctgtatctagagcgttg aatcggtgat gcgatttgtt aattaaattg atggtgtcac 180 cattaccaggtctagatata ccaatggcaa actgagcaca acaataccag tccggatcaa 240 ctggcaccatctctcccgta gtctcatcta atttttcttc cggatgaggt tccagatata 300 ccgcaacacctttattatgg tttccctgag ggaataatag aatgtcccat tcgaaatcac 360 caattctaaacctgggcgaa ttgtatttcg ggtttgttaa ctcgttccag tcaggaatgt 420 tccacgtgaagctatcttcc agcaaagtct ccacttcttc atcaaattgt ggagaatact 480 cccaatgctcttatctatgg gacttccggg aaacacagta ccgatacttc ccaattcgtc 540 ttcagagctcattgtttgtt tgaagagact aatcaaagaa tcgttttctc aaaaaaatta 600 atatcttaactgatagtttg atcaaagggg caaaacgtag gggcaaacaa acggaaaaat 660 cgtttctcaaattttctgat gccaagaact ctaaccagtc ttatctaaaa attgccttat 720 gatccgtctctccggttaca gcctgtgtaa ctgattaatc ctgcctttct aatcaccatt 780 ctaatgttttaattaaggga ttttgtcttc attaacggct ttcgctcata aaaatgttat 840 gacgttttgcccgcaggcgg gaaaccatcc acttcacgag actgatctcc tctgccggaa 900 caccgggcatctccaactta taagttggag aaataagaga atttcagatt gagagaatga 960 aaaaaaaaaaaaaaaaaaag gcagaggaga gcatagaaat ggggttcact ttttggtaaa 1020 gctatagcatgcctatcaca tataaataga gtgccagtag cgactttttt cacactcgaa 1080 atactcttactactgctctc ttgttgtttt tatcacttct tgtttcttct tggtaaatag 1140 aatatcaagctacaaaaagc atacaatcaa ctatcaacta ttaactatat cgtaatacac 1200 caagctcgacctcgcgatga gatttccttc aatttttact gcagttttat tcgcagcatc 1260 ctccgcattagctgctccag tcaacactac aacagaagat gaaacggcac aaattccggc 1320 tgaagctgtcatcggttact tagatttaga aggggatttc gatgttgctg ttttgccatt 1380 ttccaacagcacaaataacg ggttattgtt tataaatact actattgcca gcattgctgc 1440 taaagaagaaggggtacctt tggataaaag acaccaccac caccaccacc accaccacca 1500 ctcttctggtcacatcgacg acgacgacaa gttcttgggt aaaattgcta agaaccctga 1560 agccagtatgaatgtgagtc agatgatttc cttctggggc tacccaagtg agatgcataa 1620 agttataactgcagatggct atatccttca ggtctatcgg attcctcatg gaaagaatga 1680 tgctaatcatttaggtcaga gacctgttgt gtttctgcag catggtcttc ttgcctcagc 1740 tacaaactggatttccaacc ttcccaacaa cagcctgggc ttcctcctgg cagatgctgg 1800 ttatgacgtgtggctgggga acagcagagg aaacacttgg gcccaggaac atttatacta 1860 ttcaccagactcccctgaat tctgggcttt cagctttgat gaaatggctg aatatgacct 1920 tccatctacaattgatttca tcttaaagag aacaggacag aagaagctac actatgttgg 1980 ccattcccaaggcaccacca ttggttttgt cgccttttct accaatccca cactggctga 2040 aaaaatcgaagtcttccatg cattagcccc agtcgccaca gtgaagcaca cccagagcct 2100 gtttaacaaacttgcactta ttcctcactt cctcttcaag attatatttg gtaacaaaat 2160 gttctacccacacaattttt ttgaacaatt tcttggtgtt gaagtgtgct ctcgtgagac 2220 actggatgtcctttgtaaga atgccttgtt tgccattact ggagctgaca ataaaaactt 2280 caacatgagtcgcttagatg tgtatgtagc acataatcca gcaggagctt ctgttcaaaa 2340 catcctccactggagacagg ctattaagtc tgggaaattc caagcttttg actggggagc 2400 ctcagttgagaacctaatgc attataatca gcccacacct cccatctaca atttaacagc 2460 catgaatgtcccaattgcag tatggagtgc tggccaagac ctgttggctg accctcagga 2520 tgttgaccttttgctttcaa aactctctaa tctcattcac cacaaggaaa ttccaaatta 2580 caatcatctggactttatct gggcaatgga tgcacctcaa gaagtttaca atgaaattat 2640 ttctttgatggcaaaagaca aaaagtagta agcggccgct gatccgtcga gcgtcccaaa 2700 accttctcaagcaaggtttt cagtataatg ttacatgcgt acacgcgtct gtacagaaaa 2760 aaaagaaaaatttgaaatat aaataacgtt cttaatacta acataactat aaaaaaataa 2820 atagggacctagacttcagg ttgtctaact ccttcctttt cggttagagc ggatgtgggg 2880 ggagggcgtgaatgtaagcg tgacataact aattacatga tatcgacctg cagccaagct 2940 ttgaagaaaaatgcgcctta ttcaatcttt gctataaaaa atggcccaaa atctcacatt 3000 ggaagacatttgatgacctc atttctttca atgaagggcc taacggagtt gactaatgtt 3060 gtgggaaattggagcgataa gcgtgcttct gccgtggcca ggacaacgta tactcatcag 3120 ataacagcaatacctgatca ctacttcgca ctagtttctc ggtactatgc atatgatcca 3180 atatcaaaggaaatgatagc attgaaggat gagactaatc caattgagga gtggcagcat 3240 atagaacagctaaagggtag tgctgaagga agcatacgat accccgcatg gaatgggata 3300 atatcacaggaggtactaga ctacctttca tcctacataa atagacgcat ataagtacgc 3360 atttaagcataaacacgcac tatgccgttc ttctcatgta tatatatata caggcaacac 3420 gcagatataggtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt tgcattttcg 3480 gaagcgctcgttttcggaaa cgctttgaag ttcctattcc gaagttccta ttctctagaa 3540 agtataggaacttcagagcg cttttgaaaa ccaaaagcgc tctgaagacg cactttcaaa 3600 aaaccaaaaacgcaccggac tgtaacgagc tactaaaata ttgcgaatac cgcttccaca 3660 aacattgctcaaaagtatct ctttgctata tatctctgtg ctatatccct atataaccta 3720 cccatccacctttcgctcct tgaacttgca tctaaactcg acctctacat tttttatgtt 3780 tatctctagtattactcttt agacaaaaaa attgtagtaa gaactattca tagagtgaat 3840 cgaaaacaatacgaaaatgt aaacatttcc tatacgtagt atatagagac aaaatagaag 3900 aaaccgttcataattttctg accaatgaag aatcatcaac gctatcactt tctgttcaca 3960 aagtatgcgcaatccacatc ggtatagaat ataatcgggg atgcctttat cttgaaaaaa 4020 tgcacccgcagcttcgctag taatcagtaa acgcgggaag tggagtcagg ctttttttat 4080 ggaagagaaaatagacacca aagtagcctt cttctaacct taacggacct acagtgcaaa 4140 aagttatcaagagactgcat tatagagcgc acaaaggaga aaaaaagtaa tctaagatgc 4200 tttgttagaaaaatagcgct ctcgggatgc atttttgtag aacaaaaaag aagtatagat 4260 tctttgttggtaaaatagcg ctctcgcgtt gcatttctgt tctgtaaaaa tgcagctcag 4320 attctttgtttgaaaaatta gcgctctcgc gttgcatttt tgttttacaa aaatgaagca 4380 cagattcttcgttggtaaaa tagcgctttc gcgttgcatt tctgttctgt aaaaatgcag 4440 ctcagattctttgtttgaaa aattagcgct ctcgcgttgc atttttgttc tacaaaatga 4500 agcacagatgcttcgttaac aaagatatgc tattgaagtg caagatggaa acgcagaaaa 4560 tgaaccggggatgcgacgtg caagattacc tatgcaatag atgcaatagt ttctccagga 4620 accgaaatacatacattgtc ttccgtaaag cgctagacta tatattatta tacaggttca 4680 aatatactatctgtttcagg gaaaactccc aggttcggat gttcaaaatt caatgatggg 4740 taacaagtacgatcgtaaat ctgtaaaaca gtttgtcgga tattaggctg tatctcctca 4800 aagcgtattcgaatatcatt gagaagctgc tgcaggcaag tgcacaaaca atacttaaat 4860 aaatactactcagtaataac ctatttctta gcatttttga cgaaatttgc tattttgtta 4920 gagtcttttacaccatttgt ctccacacct ccgcttacat caacaccaat aacgccattt 4980 aatctaagcgcatcaccaac attttctggc gtcagtccac cagctaacat aaaatgtaag 5040 ctttcggggctctcttgcct tccaacccag tcagaaatcg agttccaatc caaaagttca 5100 cctgtcccacctgcttctga atcaaacaag ggaataaacg aatgaggttt ctgtgaagct 5160 gcactgagtagtatgttgca gtcttttgga aatacgagtc ttttaataac tggcaaaccg 5220 aggaactcttggtattcttg ccacgactca tctccatgca gttggacgat atcaatgccg 5280 taatcattgaccagagccaa aacatcctcc ttaggttgat tacgaaacac gccaaccaag 5340 tatttcggagtgcctgaact atttttatat gcttttacaa gacttgaaat tttccttgca 5400 ataaccgggtcaattgttct ctttctattg ggcacacata taatacccag caagtcagca 5460 tcggaatctagagcacattc tgcggcctct gtgctctgca agccgcaaac tttcaccaat 5520 ggaccagaactacctgtgaa attaataaca gacatactcc aagctgcctt tgtgtgctta 5580 atcacgtatactcacgtgct caatagtcac caatgccctc cctcttggcc ctctcctttt 5640 cttttttcgaccgaattaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat 5700 aggttaatgtcatgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 5760 tgcgcggaacccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 5820 gacaataaccctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 5880 atttccgtgtcgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 5940 cagaaacgctggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 6000 tcgaactggatctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 6060 caatgatgagcacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg 6120 ggcaagagcaactcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 6180 cagtcacagaaaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 6240 taaccatgagtgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 6300 agctaaccgcttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 6360 cggagctgaatgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg 6420 caacaacgttgcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 6480 taatagactggatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 6540 ctggctggtttattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 6600 cagcactggggccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 6660 aggcaactatggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 6720 attggtaactgtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 6780 tttaatttaaaaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 6840 aacgtgagttttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6900 gagatcctttttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6960 cggtggtttgtttgccggat caagagctac caactctttt tccgaaggta actggcttca 7020 gcagagcgcagataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 7080 agaactctgtagcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 7140 ccagtggcgataagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 7200 cgcagcggtcgggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 7260 acaccgaactgagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 7320 gaaaggcggacaggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 7380 ttccagggggaaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 7440 agcgtcgatttttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 7500 cggcctttttacggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 7560 tatcccctgattctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 7620 gcagccgaacgaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc 7680 ggtattttctccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta 7740 caatctgctctgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 7800 cagatcctgacgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 7860 gcgtgaccgctacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 7920 ttctcgccacgttcgccggc tttccccgtc aagctctaaa tcggggcatc cctttagggt 7980 tccgatttagtgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 8040 gtagtgggccatcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 8100 ttaatagtggactcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 8160 ttgatttataagggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 8220 aaaaatttaacgcgaatttt aacaaaatat taacaaaata ttaacgttta caggatctga 8280 attaattctattgagaagat ttaaaggtat ttgacagtag atca 8324 6 20 PRT Artificial SequenceDescription of Artificial Sequence A modified polyHis-enterokinasepolypeptide sequence 6 His His His His His His His His His His Ser SerGly His Ile Asp 1 5 10 15 Asp Asp Asp Lys 20 7 60 DNA ArtificialSequence Description of Artificial Sequence A modifiedpolyHis-enterokinase coding nucleic acid sequence 7 caccaccaccaccaccacca ccaccaccac tcttctggtc acatcgacga cgacgacaag 60

The claimed invention is:
 1. An isolated kid goat pregastric esterasewhich comprises SEQ ID NO: 2 and is free of other kid goat proteins. 2.The isolated kid goat pregastric esterase of claim 1 produced bypurifying the kid goat pregastric esterase from kid goat gullet.
 3. Theisolated kid goat pregastric esterase of claim 1 produced by recombinantgenetic expression in a non-kid goat cell.
 4. The isolated kid goatpregastric esterase of claim 3, wherein the non-kid goat cell is abacterial, a fungal, a yeast or an animal cell.
 5. The isolated kid goatpregastric esterase of claim 4, wherein the cell is a yeast cell.
 6. Theisolated kid goat pregastric esterase of claim 5 wherein the yeast isSaccharomyces cerevisiae.
 7. The isolated kid goat pregastric esteraseof claim 4, wherein the bacteria is E. Coli.
 8. The isolated kid goatpregastric esterase of claim 4, wherein the animal cell is a ChineseHamster Ovary cell.
 9. The isolated kid goat pregastric esterase ofclaim 1, wherein the isolated kid goat pregastric esterase isunaccompanied by glycosylation of kid goat pregastric esterase producedin a kid goat cell.
 10. A composition comprising a pharmaceuticallyacceptable carrier and the kid goat pregastric esterase of claim 1.